Project Summary Collectively, extracellular matrix, integrins, and Notch regulate a host of normal and pathological cellular activities. Evidence emerging from our preliminary studies shows that these cellular entities are coordinated into a signaling mechanism that has not been previously observed. The implications of our observation are broad and likely to have deep impacts on our understanding of cell interactions with cellular microenvironments as well as cellular behaviors in a range of normal and pathological scenarios. In this renewal application, the main objectives are to fill in the gaps of our current understanding of the molecular mechanism by which integrins regulate Notch and to explore the importance of this novel signaling system to several aspects of endothelial cellular function. To investigate the molecular mechanism in aim 1, we will focus on understanding how downstream integrin signaling through Src kinase impacts Notch activity. We will specifically focus on understanding how Src kinase controls 1a) the half-life of active N1ICD fragments, 1b) transcriptional activity of N1ICD, and 1c) the N1ICD transcription factor complex. In aim 2 we will determine if this signaling system is operant in a variety of endothelial functions including 2a) response to non-canonical ?3 ligands, 2b) response to matrix stiffness, and 2c) response to shear stress. Given that each of these important endothelial functions are known to be individually influenced by extracellular matrix, integrin activity, and Notch signaling, we hypothesize that these endothelial functions will also be dependent on coordinated activity between these signaling mechanisms. Throughout these studies, we will engage high school, undergraduate, and graduate students to collectively build scientific confidence and teach skills these students will require in order to pursue careers in science. At the conclusion of our studies, we will have accomplished two important milestones towards understanding this novel regulatory mechanism. Specifically, we will have unraveled many molecular details describing how integrins control Notch, and we will have defined the importance of this signaling cascade to basic endothelial cell functions which when aberrant, are associated with a significant number of human pathologies.